Caspase-mediated Bak activation and cytochrome c release during intrinsic apoptotic cell death in Jurkat cells.
Mitochondrial outer membrane permeabilization and the release of intermembrane space proteins, such as cytochrome c, are early events during intrinsic (mitochondria-mediated) apoptotic signaling. Although this process is generally accepted to require the activation of Bak or Bax, the underlying mechanism responsible for their activation during true intrinsic apoptosis is not well understood. In the current study, we investigated the molecular requirements necessary for Bak activation using distinct clones of Bax-deficient Jurkat T-lymphocytes in which the intrinsic pathway had been inhibited. Cells stably overexpressing Bcl-2/Bcl-x(L) or stably depleted of Apaf-1 were equally resistant to apoptosis induced by the DNA-damaging anticancer drug etoposide as determined by phosphatidylserine externalization and caspase activation. Strikingly, characterization of mitochondrial apoptotic events in all three drug-resistant cell lines revealed that, without exception, resistance to apoptosis was associated with an absence of Bak activation, cytochrome c release, and mitochondrial membrane depolarization. Furthermore, we found that etoposide-induced apoptosis and mitochondrial events were inhibited in cells stably overexpressing either full-length X-linked inhibitor of apoptosis protein (XIAP) or the BIR1/BIR2 domains of XIAP. Combined, our findings suggest that caspase-mediated positive amplification of initial mitochondrial changes can determine the threshold for irreversible activation of the intrinsic apoptotic pathway.
Shawgo
Mary E
ME
Department of Pharmacology, University of Kansas Medical Center, Kansas City, Kansas 66160, USA.
Shelton
Shary N
SN
Robertson
John D
JD
D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D053477:Apoptotic Protease-Activating Factor 1 / Q000235:genetics / Q000378:metabolism; D020169:Caspases / Q000235:genetics / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000235:genetics; D045304:Cytochromes c / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics; D019008:Drug Resistance, Neoplasm / Q000187:drug effects / Q000235:genetics; D005047:Etoposide / Q000494:pharmacology; D006801:Humans; D019169:Jurkat Cells; D051336:Mitochondrial Membranes / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000235:genetics / Q000378:metabolism; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000235:genetics / Q000378:metabolism*; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism; D051020:bcl-X Protein / Q000235:genetics / Q000378:metabolism
C506592:APAF1 protein, human; D000972:Antineoplastic Agents, Phytogenic; D053477:Apoptotic Protease-Activating Factor 1; C494772:BAK1 protein, human; C494826:BAX protein, human; C494811:BCL2L1 protein, human; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D005047:Etoposide; D045304:Cytochromes c; D020169:Caspases